Method for quantitative determination of renin activity in blood employing phenyl methyl sulfonyl fluoride and polyethylene glycol

ABSTRACT

A method for the quantitative determination of renin activity in blood utilizing the measurement of Angiotensin I by treating the blood with ethylenediaminetetraacetic acid (EDTA) and the plasma with phenylmethyl sulfonylfluoride (PMSF) at a preferred pH to ascertain the full range of concentration where PMSF is effective for accurate and quick determination. 
     Also, a method for the quantitative determination of renin activity in blood utilizing the measurement of Angiotensin I by incubating the samples after antibody addition at room temperature (23° to 30° C.) for 1 to 2 hours and separating the free from the antibody bound species with polyethylene glycol after having treated the blood with ethylenediaminetetraacetic acid (EDTA) and the plasma with phenylmethyl sulfonylfluoride (PMSF) at a preferred pH. PMSF offering enough protection against Angiotensin I destruction at room temperature that in combination with polyethylene glycol at a preferred pH the time required for the radioimmunoassay determination is 1 to 2 hours at room temperature instead of 24±2 hours at 4° C. which takes with charcoal in the presence of other inhibitors and making the system less succeptible to time dependent errors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-In-Part of my application entitled,Method and Reagent for Quantitative Determination of Renin Activity inBlood, Ser. No. 419,623, filed Nov. 28, 1973, now U.S. Pat. No.3,919,407, issued Nov. 11, 1975.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The measurement of plasma renin activity (PRA) is considered of primaryimportance in the investigation of hypertensive conditions. For example,it is widely employed to determine whether hypertension is due toprimary aldosteronism or secondary aldosteronism. Both conditions reveala high aldosterone secretion rate; primary aldosteronism occuring with alow renin activity, secondary aldosteronism exhibiting a high reninactivity. It is, then, important to distinguish between low renin valuesand the lower part of the normal spectrum. In other words, conditionsunder which plasma renin activity are measured must be maximized todifferentiate truly low renin values from the low but normal values.Most commercial kits on the market that determine Angiotensin I as ameasure of PRA have neglected this consideration and, as a result,cannot differentiate between truly low renin values and low but normalvalues.

Plasma renin activity (PRA) is generally measured by the quantitativedetermination of Angiotensin I. Angiotensin I can be lost after it hasbeen produced if it is not effectively inhibited as can be seen from themultireaction system shown below.

    ______________________________________                                         (1)                                                                               Angiotensinogen                                                                            ##STR1##       Angiotensin I                                 (2)                                                                               Angiotensin I                                                                              ##STR2##       Angiotensin II + Dipeptide                    (3)                                                                               Angiotensin II                                                                             ##STR3##       Inactive Peptides                            ______________________________________                                    

It is of fundamental importance then to have one or more compoundspresent in this system, which will serve as inhibitors of the sidereactions and will therefore prevent the loss of Angiotensin I.

In addition, the measurement of Angiotensin I is now assuming importancein the study of platelet-dependent thrombotic phenomena. A stimulationof immunoreactive material resembling prostaglandin E by Angiotensin IIhas been reported. Prostaglandin secretion by endothelium appears toexercise a significant role in platelet-dependent thrombotic phenomenaand in local control of vascular permeability as reported by Gimbroneand Alexander. Angiotensin II may be directly monitored by itsprecursors Angiotensin I. Obviously, an accurate and rapid method willmeet a presently growing need for Angiotensin I monitoring in theclinical patient.

2. Description of the Prior Art

I have found in my application Ser. No. 419,628, filed Nov. 28, 1973,that using phenylmethyl sulfonylfluoride (PMSF) at a final concentrationof 1.32 mg/ml plasma and adjusting the pH of the plasma to pH 6.0 theyield of Angiotensin I measured is optimized when compared withHydroxyquinoline and Dimercaprol or with Diisopropylfluorophosphate(DFP) for which clinically normal values have been established. In thismethod the addition of PMSF was such as to make the sample dilutionnegligible.

In addition, commercially available kits for the measurement of plasmarenin activity (PRA) by determination of Angiotensin I use an incubationperiod of 24±2 hours at 4° C., after the addition of antibody usingcharcoal to separate the free from the antibody bound specie in theradioimmunoassay. This long incubation at 4° C. makes it impossible toobtain results in the laboratory the same day that the sample arrives.Also, the use of charcoal to separate the free from the antibody boundspecie requires the step of carefully removing each supernatant from thepellet after centrifugation as charcoal pellets are easily disturbed.Furthermore, as charcoal is a non-specific adsorbant, the time elapsedbetween the addition of charcoal and the centrifugation of the tubes israther critical creating a real possible source of error, if manysamples are being processed.

Reports have appeared in the literature, Clinical Chemistry, by M. JamesBarrett and Patricia S. Cohen, Vol. 18, pages 1339-1342, 1972,suggesting the use of polyethylene glycol in sodium barbital buffer (25mmol/liter, pH 8.6) to separate free from antibody bound specie afterincubation of 24±2 hours at 4° C. using inhibitors which do not yieldoptimal amounts of Angiotenin I. This technique also makes it impossibleto assay the sample the same day that it is received in the laboratory.

The different methods can be summarized in the chart, which follows:

    __________________________________________________________________________    Present Method:                                                                ##STR4##                                                                     Other Methods:                                                                 ##STR5##                                                                     __________________________________________________________________________

In the above chart, the steps within the dotted circles in the presenceof PMSF as inhibitor provide the same quantitative results. These stepsare after addition of label and antibody so that the method is specificas a part of the radioimmunoassay and in no other methodology.

The advantages are as follows:

1. time of second incubation is reduced from 24±2 hours to 1 to 2 hours,allowing the test to be done the same day;

2. time dependent errors are avoided with polyethylene glycol instead ofcharcoal, as charcoal is a nonspecific adsorbant the time element afteraddition of charcoal and before centrifugation (step 7) is critical. Inother words, if one uses charcoal and doesn't centrifuge immediately, itstarts adsorbing what it should not, see article in Clinical Chemistryby Barret and Cohen; and,

3. technician's time involved in the separation of the free from thebound specie after centrifugation is substantially shortened and errorsare avoided in this separation step.

As the pellet formed after centrifugation when using polyethylene glycolis not easily disturbed, and it is this pellet in advantage 3, above,that one counts, all supernatants can be discarded in one operation bysimply inverting the rack of tubes (when tubes are firmly held by therack). The pellets remaining firmly attached to the bottom of the tubes.By contrast, the careful separation of the supernatant, which one countsin the case of charcoal, from the easily disturbed charcoal pellet(after centrifugation) requires much more careful handling of eachindividual tube and demands more of the technician's labor. This laborcan be considerable if the number of samples being processed is large.In addition, a possible error in this step is introduced unless carefulseparation is achieved.

OBJECTS OF THE INVENTION

It is the object of the present invention to ascertain the full range ofconcentrations where the inhibitor PMSF is effective for accuratedetermination and to ascertain the precise conditions for quickdetermination of Angiotensin I in plasma.

It is a further object of the present invention to provide a method,yielding the optimal amount of Angiotensin I in plasma, andsimultaneously substantially reducing the time and minimizing the timedependent errors in the assay of Angiotensin I. This technique allowsfor optimal results to be obtained the same day that the sample isreceived in the laboratory.

BRIEF SUMMARY OF THE INVENTION

In accordance with the invention, PMSF exhibits its inhibitory effectsagainst Angiotensin I destruction between 0.013 mg and 13.2 mg PMSF perml of plasma. The effectiveness of its inhibitory capacity varyingwidely between these ranges and should be taken into consideration whenaccurate and quick determination of Angiotensin I is being made.

This aspect of the invention which relates to the broad concentration ofeffectiveness of PMSF finds use in both radioactive and non-radioactivetesting.

Also, in accordance with the invention, the yield of Angiotensin I byradioimmunoassay is the same upon incubating the reaction mixture (afterantibody addition) for 1 to 2 hours at 23° to 30° C. and subsequentaddition of polyethylene glycol (12% to 18% final concentration) in 0.01M Tris hydroxymethylaminomethane (Tris) buffer pH about 7.0 or distilledwater pH 5.6 to 6.5 or when incubating the reaction mixture mixture(after antibody addition) for 24±2 hours at 4° C. and subsequentaddition of charcoal, when PMSF (0.013 mg to 13.2 mg) is used asinhibitor at a specified pH. The optimal pH being about 6.0.

This aspect of the invention relating to polyethylene glycol is uniquelyadapted for radioimmunoassay testing.

DETAILED DESCRIPTION OF THE INVENTION

The reagents used are:

1. Phenylmethyl sulfonylfluoride--(Sigma Chemical Co.) Solution of thesereagents in ethanol were made and added to plasma as to give finalconcentrations between 0.013 mg and 13.2 mg PMSF per ml of plasma.

2. Dimercaprol solution--(E. R. Squibb & Sons, Inc.) 300 mg 2-3dimercaptopropanol and 600 mg benzyl benzoate in 3 ml peanut oil.

3. 8-Hydroxyquinoline solution--(E. R. Squibb & Sons, Inc.) 660 mgdissolved in 10 ml of water.

4. Diisopropylfluorophosphate--(Sigma Chemical Co.) 0.1 mlDiisopropylfluorophosphate (DFP) in 1.9 ml isopropyl alcohol.

5. Polyethylene glycol solutions:

a. 24% to 36% polyethylene glycol (carbowax 6000 flakes, Schwarz-Mann)solutions in 0.01 M Tris hydroxymethylaminomethane (Tris) buffer at pH7.0. These solutions are referred to as 24% or 36% carbowax. As thecarbowax solutions are added to 1 ml of solution, the finalconcentration of carbowax is 12% to 18%.

b. Similar solutions of carbowax were made in distilled water. The pH ofthe water when tested with pH paper was between 5.6 and 6.5. Thesesolutions were also added to 1 ml of solution.

6. I¹²⁵ Angiotensin I, Angiotensin I standard, Angiotensin I antibody,and charcoal were purchased as a kit (E. R. Squibb & Sons, Inc.). Thesereagents were prepared and used following the manufacturers directions.

DETAILED DESCRIPTION OF THE PREFERRED METHOD EMBODIMENT

The collection of blood should be in a cold Vacutainer containing EDTA(liquid EDTA preferred), inverted several times and packed in iceimmediately. Centrifugation is carried out in the cold to collect theplasma. The plasma is kept in ice if processed within 2 hours or it canbe frozen at -10° C. until ready for use. In preparing the sample, ifthe sample has been frozen, it is allowed to thaw while immersed incrushed ice. It should be noted that from the time the plasma sample isobtained, all manipulations are carried out in the cold (4° C.), unlessotherwise specified.

Using a pH meter, the pH of the sample is adjusted to the desired pHwith 0.5 M HCl while different volumes of phenylmethyl sulfonylfluoride(PMSF) solutions are added so that the final concentration is between0.013 and 13.2 mg PMSF per ml of plasma. Other inhibitors are added toother samples for purpose of comparison. Either 10 μl of8-Hydroxyquinoline solution and 10 μl of Dimercaprol solution per ml ofplasma are added according to the manufacturer's directions or 20 μl ofDiisopropylfluorophosphate per ml of plasma according to the publishedliterature. The mixture is then vigorously mixed and divided into fouraliquots. In the incubation, one of the four aliquots per specimen, twoare placed in a shaking water bath at 37° C. for usually 3 hours. Theother two aliquots, to be used as blanks, are kept in the ice bath(about 4° C.) for the same amount of time.

In the radioimmunoassay, to four tubes containing 1 ml of diluted I¹²⁵Angiotensin I, 50 and 10 μl from each of the two aliquots incubated at37° C. are added. These two sets of different size aliquots areconsidered duplicates. To two other tubes, 50 μl are added from each ofthe two aliquots incubated at 4° C. These two tubes represent duplicateblanks. To all the tubes, 50 μl of Angiotensin I antiserum are added.After Angiotensin I antiserum is added, one of the following proceduresis followed:

a. The mixture is incubated at 4° C. for 24±2 hours. At the end of theincubation period 1.0 ml of charcoal suspension is added to all tubes.The mixture is then mixed gently and centrifuged for 5 minutes at 3,000to 4,000 rmp. (During this separation step, the charcoal suspensionshould be added only to the quantity of tubes that can be centrifugedsimultaneously as the equilibrium of the antigen-antibody reaction isaltered upon prolonged contact with the charcoal--manufacturer'sdirections). The supernatant is carefully separated from the pellet andthe radioactivity counted; or,

b. The mixture is incubated for 1 to 2 hours at room temperature 23° to30° C. The tubes are then placed at 4° C. Then, 100 μl (or up to 250 μl)of serum or any equivalent material containing similar quantities ofimmunoglobulins are added to aid in the precipitation of immunoglobulinsin the plasma followed by 1 ml of carbowax solution (24% to 36%carbowax). The tubes are left at 4° C. for 10 minutes (or 30 minutes orup to 2 hours) then centrifuged at 5,000 rpm for 15 minutes. Thesupernatant is poured off, the tubes are allowed to drain for 2 minutesand the radioactivity of the pellet counted. The pouring off of thesupernatant can be accomplished in one step by inverting the entire rackof tubes (when all the tubes are well fitted in the rack). Thesupernatant is discarded.

A standard curve is prepared by setting up tubes with known amounts ofAngiotensin I and following the same procedure used with the samples.

Table I shows the results of Angiotensin I obtained when the plasmasamples were adjusted to pH 6.0 and different concentrations of PMSFwere added. The samples were incubated, after antibody additions, for 24hours at 4° C. and charcoal was used to separate the free from the boundspecie. The results obtained usng Dimercaprol and Hydroxyquinolinetogether or DFP instead of PMSF are also shown. These other inhibitorsare shown as the clinically normal range for Angiotensin I has beenestablished using these inhibitors.

Each number shown in Table I is the average or 6 determinations. Thesame is true for Table III (shown later).

Table I shows that, as shown in our previous patent, the addition of1.32 mg PMSF per ml of plasma gives greater yield of Angiotensin I thanusing Hydroxyquinoline and Dimercaprol and as good a yield ofAngiotensin I as when treating the sample with the very toxic DFP.

When the concentrations of PMSF are varied to 0.66 mg/ml plasma (0.5times the original 1.32 mg/ml plasma), 0.132 mg/ml plasma (0.1 times theoriginal 1.32 mg/ml plasma) and 0.013 mg/ml plasma (0.01 times theoriginal 1.32 mg/ml plasma), the yield of Angiotensin I decreases by10%, 26% and 65%, respectively, when compared with 1.32 mg PMSF/ml.

On the other hand, when the concentration of PMSF is increased to 6.60mg/ml plasma (5 times the original 1.32 mg/ml plasma) and 13.2 mg/mlplasma (10 times the original 1.32 mg/ml plasma), there is first anincrease of about 30%, then a decrease of about 74%, respectively.

Thus, if an error in addition of the inhibitor PMSF is made, one must beaware of the difference in yield of Angiotensin I when comparing theresults obtained in a patient with the clinically normal values reportedfor Angiotensin I.

In addition, as when the concentration of PMSF is around 6.6 mg PMSF/mlof plasma shows a further yield of Angiotensin I, clinically normalvalues should be reestablished to maximize the yield.

The above percentages are representative of average values obtained inmy studies, but I have found that the values can vary about 10% indifferent plasma samples from different individuals.

I have used 1.32 mg PMSF/ml plasma as my base line for comparison withother PMSF concentrations, as it gives about the same values as withDFP. The clinical normal values for DFP have been established andtherefore each individual result obtained in the laboratory must becompared with these values in order to interpret the numbers obtained.The present method has proved its accuracy.

The above percentages, although obtained in a small sample population,give guidelines for making meaningful interpretations where comparingthe results obtained in the laboratory with the clinically establishedrange of results if an error of addition with the inhibitor PMSFoccurred and is taken into account.

Thus, with the above explained procedure, and using the Tables herein,it is possible to perform meaningful analysis in a situation where asecond sample of venous blood may not be possible to obtain under theprecisely controlled conditions of blood drawing from the veins of theright and left kidneys in the clinical procedure for differential renalplasma renin activity, which is an essential part of the diagnosis forsurgically correctable reno-vascular hypertension. It is now possiblefor the first time to achieve reliable clinical data even though errorshave been detected in the procedure.

For example, if one gets an average of 8.6 ng Angiotension I/ml/hr in aplasma sample to which one has added (1.32×5) mg PMSF/ml of plasma(instead of 1.32) one should be aware that for that patient the valuethat one must compare with the clinically established range is 6.0±0.6ng Antiotensin I/ml/hr and not 8.6 as the above difference ofconcentration of inhibitor PMSF gives an enhancement of 30% (±10%) inAngiotensin I. If, on the other hand, one adds by mistake (1.32×10) mgPMSF/ml of plasma (instead of 1.32) and gets 1.6 ng Angiotensin I/ml/hr,the value for that patient, which one should compare with the clinicallyestablished range, is still 6.0±0.6, and not 1.6 as this error inaddition shows a decrease of 74% (±10%) in the Angiotensin I obtained,and so on.

Also, the higher values of Angiotensin I obtained in plasma treated withabout 6.6 mg PMSF/ml of plasma may shed further light on the mechanismof inhibition of this compound.

When the samples are adjusted to pH 5.0 or 7.5, the same concentrationsof PMSF appear similarly effective in the inhibition of Angiotensin Idestruction although all results were lower than those obtained at pH6.0, which is the optimal pH for Angiotensin I production.

Table II shows the results obtained when using carbowax or charcoalunder different incubation conditions (24 hours at 4° C. with charcoaland 11/2 hours at 28° C. with carbowax). The plasma samples wereadjusted to pH 6.0 and 1.32 mg PMSF/ml or plasma were added to theplasma. As shown in this table, the results are very similar in eithercase.

Thus, by significantly decreasing the incubation time, optimal resultscan be obtained in the same day.

Similar results between the two incubation conditions are obtained whenthe samples are adjusted to pH 5.0 to 7.5 except that, all values arelower than those obtained at pH 6.0.

For latter reference, it should be pointed out here that in theexperiments shown in Table II, the samples were allowed to stand at 4°C. after carbowax addition and before centrifugation, for 10 minutes.

Table III shows the amounts of Angiotensin I obtained when theconcentration of PMSF is varied in plasma samples at pH 6.0 and carbowaxis used in the shorter incubation method. Comparison with Table I showsthat the range of concentration of PMSF in plasma, namely, 0.013 to 13.2mg PMSF/ml of plasma, follows the same pattern of effectiveness as withthe longer incubation method. As in the case of charcoal, similar butlower results are obtained at pH 5.0 or 7.5.

Table IV shows the results obtained when after addition of carbowax andbefore centrifugation, the samples are placed at 4° C. for 30 minutes.All other conditions are the same as those in Table II. Comparison ofTables II and IV show that the time elapsed between the addition ofcarbowax and centrifugation of the samples is not critical as identicalresults are obtained. The results do not vary even after 2 hours at 4°C. instead of 30 minutes.

The fact that the time elapsed is not critical (in contrast to thecharcoal methodology) allows to centrifuge together a large number ofsamples after carbowax has been added without time dependent errors.

TABLE I

Plasma renin activity using different concentrations of PMSF at pH 6.0.

Incubation conditions: 24 hours, 4° C.

Method of separation: charcoal

    ______________________________________                                                    Final Concentration                                                                         PRA                                                             of PMSF       (ng Angio % Differ-                                 Inhibitor Used                                                                            (mg/ml plasma)                                                                              I/ml/hr)  ence.sup.(1)                              ______________________________________                                        Hydroxyquinoline          1.5       35%                                       and Dimercaprol.sup.(2)                                                       DFP.sup.(2)               2.3        0%                                       PMSF        0.0132        0.8       65%                                                   (1.32 × 0.01)                                               PMSF        0.132         1.7       26%                                                   (1.32 × 0.1)                                                PMSF        0.66          2.1        9%                                                   (1.32 × 0.5)                                                PMSF        1.32          2.3                                                 PMSF        6.60          3.3       30%                                                   (1.32 × 5)                                                  PMSF        13.2          0.6       74%                                                   (1.32 × 10)                                                 ______________________________________                                         .sup.(1) % difference was obtained setting the highest of the two values      being compared to 100%                                                        .sup.(2) amounts used described under reagents                           

TABLE II

Plasma renin activity using carbowax or charcoal to separate free frombound species at different incubation conditions. Standing time at 4° C.after carbowax addition is 10 minutes.

    ______________________________________                                        PRA                                                                           ng Angiotensin I/ml/hr                                                                   15% Carbowax.sup.(1)                                                          (final concentration)                                                                       Charcoal                                             Sample     Incubation:   Incubation:                                          No.        11/2 hr at 28° C.                                                                    24 hr at 4° C.                                ______________________________________                                        1          2.2           2.0                                                  2          3.8           3.9                                                  3          0.9           0.8                                                  4          1.6           1.4                                                  5          1.7           1.9                                                  6          2.4           2.3                                                  7          1.3           1.3                                                  8          3.5           3.3                                                  ______________________________________                                         .sup.(1) 200 μl of serum added before carbowax addition               

TABLE III

Plasma renin activity using different concentrations of PMSF at pH 6.0.

Incubation conditions: 11/2 hours, 28° C.

Method of separation: carbowax (15% final concentration)

    ______________________________________                                                    Final Concentration                                                                         PRA                                                             of PMSF       (ng Angio % Differ-                                 Inhibitor Used                                                                            (mg/ml plasma)                                                                              I/ml/hr)  ence.sup.(1)                              ______________________________________                                        Hydroxyquinoline.sup.(2)  1.5       37%                                       and Dimercaprol                                                               DFP.sup.(2)               2.3        4%                                       PMSF        0.013         0.8       67%                                                   (1.32 × 0.01)                                               PMSF        0.132         1.7       29%                                                   (1.32 × 0.1)                                                PMSF        0.66          2.2        8%                                                   (1.32 × 0.5)                                                PMSF        1.32          2.4                                                 PMSF        6.60          3.4       29%                                                   (1.32 × 5)                                                  PMSF        13.2          0.6       75%                                                   (1.32 × 10)                                                 ______________________________________                                         .sup.(1) % difference obtained setting the highest of the two values bein     compared to 100%                                                              .sup.(2) amounts used described under reagents                           

TABLE IV

Plasma renin activity using carbowax or charcoal to separate free frombound species at different incubation conditions. Standing time at 4° C.after carbowax addition is 30 minutes.

    ______________________________________                                        PRA                                                                           ng Angiotensin I/ml/hr                                                                   15% Carbowax.sup.(1)                                                          (final concentration)                                                                       Charcoal                                             Sample     Incubation:   Incubation:                                          No.        11/2 hr at 28° C.                                                                    24 hr at 4° C.                                ______________________________________                                        1          2.1           2.1                                                  2          3.7           3.8                                                  3          0.9           0.8                                                  4          1.4           1.3                                                  5          1.9           1.7                                                  6          2.5           2.5                                                  7          1.4           1.2                                                  8          3.4           3.2                                                  ______________________________________                                         .sup.(1) 200 μl serum added before carbowax addition                  

In the foregoing examples, the temperature for incubation is illustratedat 28° C., but the incubation is equally effective at temperatures ofabout 23° C. to about 30° C. for a time period of about 1 hour to about2 hours, although the preference is about 2 hours.

Also the amount of serum to aid in the precipitation of immunoglobulinsin the plasma used before carbowax addition may vary from about 100 μlto about 250 μl, but preferably and for the technicians' convenience,smaller amounts should be used, e.g., about 100.

Although Tables II and IV are shown with 200 μl of serum added, the sameresults can be obtained with 100 μl.

Further, the carbowax concentration shown in Tables II and IV is about15% final concentration, but equally good results are obtained if thecarbowax is 12% to 18% (final concentration).

Also, interestingly, if the carbowax solutions were made in distilledwater rather than Tris buffer, similar standard curves and results wereobtained.

This shows that neither the particular ionic strength nor exact pH ofthe buffer is needed for the effectiveness of polyethylene glycol inseparating the free from the bound species, and that as long aspolyethylene glycol is present within the specified final concentrationrange, the buffer is not necessary.

In summary, the methodology presented here gives a faster and more errorfree method for the determination of Angiotensin I in serum. Inaddition, as the data in Tables I and III give representativepercentages obtained in our sample population, these percentages provideuseful guidelines in obtaining meaningful answers for patients' resultswhere errors in the addition of inhibitor are made.

The use of Tris buffer pH 7.0 was made originally as proteins are knownto denature in either very acid or very basic pH, therefore, ourselection of a neutral pH.

The fact that distilled water (pH 5.6-6.5) gave similar results as theabove mentioned buffer was an interesting and significant finding.

What is claimed is:
 1. In a method for measuring plasma renin activityin a sample of plasma comprising adding ethylene diamine tetraaceticacid, adding inhibitor to inhibit Angiotensin I destruction, incubatingat about 37° C. to release Angiotensin I from the plasma sample, addingI¹²⁵ labeled Angiotensin I while adding antibody, incubating the labeledmixture sample and antibody and adding a material to separate freeAngiotensin I from Angiotensin I bound to antibody prior to determiningthe amount of Angiotensin I that improvement consisting of:adding phenylmethyl sulfonyl fluoride as the inhibitor; adjusting the pH from 5 to7.5 after adding the inhibitor; incubating the labeled mixture of sampleand antibody for 1 to 2 hours at about 23° C. to 30° C. after theincubation at 37° C. to yield Angiotensin I for measurement; separatingthe free Angiotensin I from the Angiotensin I bound to antibody withpolyethylene glycol in 0.01 Molar Tris hydroxymethyl aminomethane at pH7 in a concentration of 12% to 18% based on the total medium, in thepresence of about 100 μl to 250 μl of serum or any equivalent materialcontaining similar quantities of immunoglobulins to aid precipitation ofimmunoglobulins in the plasma; and, said incubating and separating stepsbeing carried out one after the other, allowing for the optimal yield ofAngiotensin I to be obtained in the same day that the sample is receivedand minimizing time dependent errors occurring therein.
 2. A method ofmeasuring plasma renin activity in a plasma sample comprising adjustingthe plasma pH from 5 to 7.5 in the presence ofethylenediaminetetraacetic acid and in the presence of between 0.013 and13.2 mg phenylmethyl sulfonylfluoride per ml of plasma as inhibitoragainst Angiotensin I destruction, incubating at about 37° C. to releaseAngiotensin I from the plasma, adding I¹²⁵ labeled Angiotensin I whileadding antibody to Angiotensin I, thereafter, incubating to allowreaction with antibody to Angiotensin I and adding a substance toseparate the free Angiotensin I from the Angiotensin I bound to antibodyprior to determining the amount of Angiotensin I, that improvementconsisting of incubating for 1 to 2 hours at about 23° C. to 30° C. andseparating free from bound Angiotensin I, using polyethylene glycol at a12% to 18% final concentration in a pH range provided by 0.01 M trishydroxymethylaminomethane at pH 7.0 or by distilled water at pH 5.6 to6.5 in the presence of about 100 μl to 250 μl of serum or any equivalentmaterial containing similar quantities of immunoglobulins and saidincubating and separating steps being carried out one after the otherallowing for the optimal yield of Angiotensin I to be obtained in thesame day that the sample is received and minimizing time dependenterrors occurring in the separation as compared to separations usingsolid adsorbent materials. .[.3. In a method for measuring plasma reninactivity in a sample of plasma comprising addingethylenediaminetetraacetic acid while adjusting the pH of said sample,adding inhibitor to prevent Angiotensin I destruction, incubating atabout 37° C. to release Angiotensin I from the sample and thereafterdetermining the amount of Angiotensin I thus released that improvementconsisting of adding as the inhibitor phenylmethyl sulfonylfluoride in arange of concentration between 0.013 mg/ml to 13.2 mg/ml of plasmasample while simultaneously adjusting the pH to 5 to 7.5..].